Fluorescence polarization immunoassay (FPIA) is a common method of analyzing liquid samples for the presence of an analyte of interest. As described in U.S. Pat. Nos. 5,391,740, 4,939,264, 4,585,862 and 4,492,762, the salient portions of which are incorporated herein by reference, as well as elsewhere, this technique generally consists of measuring the polarization characteristics of a diluted sample of the liquid mixed with a fluorophore and a binding substance that binds the fluorophore to the analyte of interest in an amount that is dependent on the concentration of that analyte.
It has long been recognized that some endogenous substances (bilirubin, for example) may interfere with FPIA measurements due to their inherent polarization characteristics. The conventional method for eliminating this interference is to measure the polarization characteristics of a "blank." That is, the polarization characteristics are measured for a mixture of sample, reagents and diluent, but without the fluorophore, at a sample dilution that is identical to the dilution of the sample mixture to be measured after incubation with the reagents (including the fluorophore and binding substance). The polarization characteristics of the "blank" are subtracted from the polarization characteristics of the actual sample, yielding the polarization level of the analyte/fluorophore mixture free of the interference of other constituents in the sample (Jolley et. al, Clin Chem 27 (7):1189-1197, 1981).
The measurement of a blank is easily executed in manual and small immunoassay systems. One method is to perform two analyses in two cuvettes, the difference being that one of the analyses is the blank and does not contain the fluorophore in its reagent mixture. A second method is to perform the two analyses sequentially. The sample and reagents (without the fluorophore) are mixed in a cuvette and the blank polarization characteristics are measured. Additional sample is then deposited in the cuvette along with additional reagents (maintaining the same sample dilution as in the blank measurement) and, following incubation, the final polarization characteristics are measured. In both of these methods, as in all current FPIA analysis systems, the dilution of the sample is the same in both the blank read and the final read of the polarization characteristics.
These methods of measuring blank polarization characteristics can be somewhat problematical for high-volume automated immunoassay systems, such as those performed on devices similar to that described in U.S. Pat. No. 5,358,691, incorporated herein by reference. The first method requires two analysis cuvettes. With a system that processes cuvettes in an assembly-line fashion, this requirement effectively reduces the processing capacity of the system by fifty percent, since every other cuvette is used for blank measurement. The second method requires only one cuvette, but it requires two separate depositions of sample into that cuvette. Again, for an automated system that operates in an assembly-line fashion, this requirement excessively complicates system design and manufacture. The second addition of sample necessarily takes place at a later time than the first deposition, since the reading of the blank polarization characteristics must be interposed. In an assembly-line instrument, this difference in time means that the second deposition of sample must occur at a different location as well, so the sample must be transported or piped to two separate locations for deposition in the analysis cuvette. This dual sample access requirement increases instrument complexity and cost, reduces instrument reliability, and presents increased risk of sample spillage and instrument contamination. There is, therefore, great utility for a method of performing FPIA analytical tests that requires only a single sample access during the analytical process.